Suppressive effect of pioglitazone, a PPAR gamma ligand, on azoxymethane-induced colon aberrant crypt foci in KK-Ay mice.

Obesity is an established risk factor for colorectal cancer. Pioglitazone is a peroxisome proliferator- activated receptor γ(PPARγ) agonist that induces differentiation in adipocytes and induces growth arrest and/or apoptosis in vitro in several cancer cell lines. In the present study, we investigated the effect of pioglitazone on the development of azoxymethane-induced colon aberrant crypt foci (ACF) in KK-Ay obesity and diabetes model mice, and tried to clarify mechanisms by which the PPARγ ligand inhibits ACF development. Administration of 800 ppm pioglitazone reduced the number of colon ACF / mouse to 30% of those in untreated mice and improved hypertrophic changes of adipocytes in KK-Ay mice with significant reduction of serum triglyceride and insulin levels. Moreover, mRNA levels of adipocytokines, such as leptin, monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1, in the visceral fat were decreased. PCNA immunohistochemistry revealed that pioglitazone treatment suppressed cell proliferation in the colorectal epithelium with elevation of p27 and p53 gene expression. These results suggest that pioglitazone prevented obesity-associated colon carcinogenesis through improvement of dysregulated adipocytokine levels and high serum levels of triglyceride and insulin, and increase of p27 and p53 mRNA levels in the colorectal mucosa. These data indicate that pioglitazone warrants attention as a potential chemopreventive agent against obesity-associated colorectal cancer.

Loss of adiponectin promotes intestinal carcinogenesis in Min and wild-type mice.

BACKGROUND & AIMS: Metabolic syndrome- and obesity-associated cancers, including colon cancer, are common in Western countries. Visceral fat accumulation and decreased levels of plasma adiponectin (APN) have been associated with development of human colorectal adenoma. We investigated the function of APN in intestinal carcinogenesis. METHODS: APN+/+, APN+/-, or APN-/- mice (C57BL/6J) were given injections of azoxymethane (AOM), which led to development of intestinal tumors; these strains of mice were also crossed with Min mice to assess polyp formation. Adipocytokine levels and phosphorylation/activation of AMP-activated protein kinase (AMPK) were evaluated to investigate the mechanisms of APN in tumor growth. RESULTS: The total number of polyps in the intestines of male APN+/-Min and APN-/-Min mice increased 2.4- and 3.2-fold, respectively, by the age of 9 weeks and 3.2- and 3.4-fold, respectively, by 12 weeks, compared with those of APN+/+Min mice. Similar results were obtained from female mice. AOM induced colon tumor formation in 40% of APN+/+, 50% of APN+/-, and 71% of APN-/- (P<.05) mice, respectively; mean values for tumor multiplicity of each genotype were 0.5, 0.6, and 1.1 (P<.05), respectively. Phosphorylation of AMPK decreased in intestinal epithelial cells of APN-/- mice compared with APN+/+ mice. Among serum adipocytokines, plasminogen activator inhibitor-1 levels increased in APN-/-Min mice and APN-/- mice that received injections of AOM. Activation of AMPK suppressed expression of plasminogen activator inhibitor-1 in Min mice. CONCLUSIONS: Mice with disruptions in APN develop more intestinal tumors and have decreased activation (phosphorylation) of AMPK and increased levels of plasminogen activator inhibitor-1, compared with wild-type mice. APN and its receptor might be developed as targets for cancer chemopreventive agents.

Inhibition of intestinal polyp formation by pitavastatin, a HMG-CoA reductase inhibitor.

It has been suggested that hyperlipidemia is positively associated with colon carcinogenesis. Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors, reduce serum lipid levels. In this study, we clarified the effects of a novel chemically synthesized statin, pitavastatin, on intestinal polyp formation in Min mice, and further examined serum lipid and adipocytokine levels, and proinflammatory and adipocytokine gene levels in intestinal mucosa of Min mice. Treatment with pitavastatin at doses of 20 and 40 ppm decreased the total number of polyps dose-dependently to 85.2% and 65.8% (P < 0.05) of the untreated value, respectively. Serum levels of total cholesterol and triglyceride were slightly reduced and those of IL-6, leptin, and MCP-1 were decreased by 40-ppm pitavastatin treatment. mRNA expression levels of cyclooxygenase-2, IL-6, inducible nitric oxide (iNOS), MCP-1, and Pai-1 were significantly reduced in intestinal nonpolyp parts by pitavastatin treatment. Among them, iNOS mRNA levels were also reduced in the intestinal polyps. Moreover, oxidative stress represented by 8-nitroguanosine in the small intestinal epithelial cells was reduced by pitavastatin treatment. Related to these proinflammatory genes, PPARγ activity was activated in the intestinal nonpolyp parts and in the liver of Min mice with pitavastatin treatment. These results indicated that pitavastatin has potential benefit for the suppression of intestinal polyp development.

High susceptibility to azoxymethane-induced colorectal carcinogenesis in obese KK-Ay mice.

Obesity is associated with colon carcinogenesis. However, not much information is available regarding the mechanisms of obesity-associated colorectal cancer, and there are only few useful animal models for investigating the underlying mechanism between obesity and colorectal cancer. KK-A(y) mice exhibit severe obesity. Amount of visceral fat assessed by micro-computed tomography was almost 15 times higher than that of same aged C57BL/6J mice. Treatment with azoxymethane (AOM; 200 µg/mouse injected once a week for 3 times) resulted in markedly increased colon aberrant crypt foci (ACF) development (≈70 ACF/mouse) in KK-A(y) mice compared with lean C57BL/6J mice (≈9 ACF/mouse). Moreover, administration of AOM at a dose of 200 µg/mouse once a week for 6 times developed colorectal adenocarcinomas within only 7 weeks after the last AOM injection. The incidence of adenocarcinoma was 88% in KK-A(y) mice and was markedly higher than the 4% observed in C57BL/6J mice. The number of tumors/mouse was 7.80 in KK-A(y) mice and also markedly higher than the 0.12 in the C57BL/6J case. Interestingly, adenocarcinomas were observed in most of the AOM-treated KK-A(y) mice along with remarkable tumor angiogenesis, and some showed submucosal invasion. These results indicate that the KK-A(y) mouse, featuring intact leptin and leptin receptor Ob-Rbl, could be a useful animal model to investigate obesity-associated cancer.

Increase of oxidant-related triglycerides and phosphatidylcholines in serum and small intestinal mucosa during development of intestinal polyp formation in Min mice.

Recent epidemiological studies have shown a positive association of a high-fat diet with the risk of colon cancer. Indeed, increments in the serum levels of triglycerides (TG) and cholesterols are positively related with colon carcinogenesis. We previously reported that an age-dependent hyperlipidemic state is characteristic of Min mice, an animal model for human familial adenomatous polyposis (FAP). However, qualitative and quantitative changes of lipid metabolism are poorly understood in this state. Here, we provide detailed analysis of serum lipids in Min mice using reverse-phased liquid chromatography/electrospray ionization mass spectrometry (RPLC/ESI-MS). We also demonstrate local analysis of lipid droplets in the villi of the small intestine using laser capture microdissection and a sensitive chip-based nanoESI-MS system. As a result, oxidized phosphatidylcholines (PC) such as aldehyde and carboxylic acid types were increased, even at an early stage of intestinal polyp formation in serum. In addition, hydroperoxidizable TG precursors containing linoleic acid (18:2n-6) were deposited at the tip of the villi with aging, and these hydroperoxidized TG were also increased in serum. Meanwhile, increments of the oxidizable TG precursors in serum and small intestinal mucosa were suppressed by treatment with pitavastatin, a novel third generation lipophilic statin. These results suggest that quantitative and qualitative lipid changes such as hydroperoxidizable TG precursors are important in the course of intestinal polyp formation and oxidative stress might lead to the development of intestinal polyp formation in Min mice.

Oral supplementation with dihomo-gamma-linolenic acid-enriched oil altered serum fatty acids in healthy men.

Dihomo-gamma-linolenic acid (DGLA)-enriched oil (50 or 150 mg as free DGLA) was administered to healthy men for 4 weeks. The DGLA content in serum phospholipids dose-dependently increased and returned to the initial level after a 4-week washout. No side effects or changes in platelet aggregation were observed. These results indicate that oral supplementation with DGLA oil can safely increase serum DGLA content.

Overexpression of low-density lipoprotein receptor and lipid accumulation in intestinal polyps in Min mice.

Apc-deficient Min mice feature low expression of lipoprotein lipase (LPL), high concentration of serum triglyceride (TG), fatty change of the liver and large numbers of intestinal polyps. We have reported that induction of LPL expression reduces serum lipid, especially TG, improves fatty change of the liver and inhibits intestinal polyp formation in the mice. In this study, fatty change/lipid accumulation in intestinal mucosa and polyps in Min mice were analyzed by Oil-red O staining and electron microscopy. A number of large lipid droplets were found in the epithelia of the upper part of polyps. On the other hand, small lipid droplets were only slightly observed at the tip of the villi in non-tumoros parts of the small intestine of Min mice and in the villi of wild-type mice. Moreover, low-density lipoprotein receptor (LDLR) was overexpressed in the area where lipid droplets were observed. The expression levels of LDLR mRNA in the intestinal polyps of Min mice were approximately 3 times higher compared to those in the non-tumoros parts. Remarkable expression of cyclooxygenase-2 was mainly distributed in stromal cells and some in epithelial cells. It is speculated that lipid accumulation in the intestinal polyps may play an important role in intestinal polyp formation in Apc-deficient mice.